Molecular biology and pharmaceutical drug development now make intensive use of nucleic acid analysis. The most challenging areas are whole genome sequencing, single nucleotide polymorphism detection, screening and gene expression monitoring.
One area of technology which has improved the study of nucleic acids is the development of fabricated arrays of immobilised nucleic acids. These arrays typically consist of a high-density matrix of polynucleotides immobilised onto a solid support material. Fodor et al., Trends in Biotechnology (1994) 12:19-26, describe ways of assembling the nucleic acid arrays using a chemically sensitised glass surface protected by a mask, but exposed at defined areas to allow attachment of suitably modified nucleotides. Typically, these arrays may be described as “many molecule” arrays, as distinct regions are formed on the solid support comprising a high density of one specific type of polynucleotide.
An alternative approach is described by Schena et al., Science (1995) 270:467-470, where samples of DNA are positioned at predetermined sites on a glass microscope slide by robotic micropipetting techniques.
WO 98/44151 and WO 00/18957 both describe methods of forming polynucleotide arrays based on “solid-phase” nucleic acid amplification, which is analogous to a polymerase chain reaction wherein the amplification products are immobilised on a solid support in order to form arrays comprised of nucleic acid clusters or “colonies”. Each cluster or colony on such an array is formed from a plurality of identical immobilised polynucleotide strands and a plurality of identical immobilised complementary polynucleotide strands. The arrays so-formed are generally referred to herein as “clustered arrays” and their general features will be further understood by reference to WO 98/44151 or WO 00/18957, the contents of both documents being incorporated herein in their entirety by reference.
As aforesaid, the solid-phase amplification methods of WO 98/44151 and WO 00/18957 are essentially a form of the polymerase chain reaction carried out on a solid support. Like any nucleic acid amplification reaction these methods require the use of forward and reverse amplification primers capable of annealing to a template to be amplified. In the methods of WO 98/44151 and WO 00/18957 both primers are immobilised on the solid support at the 5′ end. Other forms of solid-phase amplification are known in which only one primer is immobilised and the other is present in free solution (Mitra, R. D and Church, G. M., Nucleic Acids Research, 1999, Vol. 27, e34; Shendure J, Porreca G. J., Reppas N. B. et al, Science, 2005, Vol. 309, 5741, 1728-1732; Margulies M., Egholm M., Altman W. E. et al, Nature, 2005, 437, 376-380).
In common with all nucleic acid amplification techniques, solid-phase amplification requires the use of forward and reverse amplification primers which include “template-specific” nucleotide sequences which are capable of annealing to sequences in the template to be amplified, or the complement thereof, under the conditions of the annealing steps of the amplification reaction. The sequences in the template to which the primers anneal under conditions of the amplification reaction may be referred to herein as “primer-binding” sequences.
The amplification reaction cannot occur in the absence of annealing of the forward and reverse primers to primer binding sequences in the template to be amplified under the conditions of the annealing steps of the amplification reaction, i.e. if there is insufficient complementarity between primers and template. Some prior knowledge of the sequence of the template is therefore required before one can carry out an amplification reaction to amplify a specific template. The user must usually know the sequence of at least the primer-binding sites in the template in advance so that appropriate primers can be designed, although the remaining sequence of the template may be unknown. The need for prior knowledge of the sequence of the template increases the complexity and cost of solid phase amplification of complex mixtures of templates, such as genomic DNA fragments.
Certain embodiments of the methods described in WO 98/44151 and WO 00/18957 make use of “universal” primers to amplify templates comprising a variable template portion that it is desired to amplify flanked 5′ and 3′ by common or “universal” primer binding sequences. The “universal” forward and reverse primers include sequences capable of annealing to the “universal” primer binding sequences in the template construct. The variable template or target) portion may itself be of known, unknown or partially known sequence. This approach has the advantage that it is not necessary to design a specific pair of primers for each template to be amplified; the same primers can be used for amplification of different templates provided that each template is modified by addition of the same universal primer-binding sequences to its 5′ and 3′ ends. The variable template sequence can therefore be any DNA fragment of interest. An analogous approach can be used to amplify a mixture of templates, such as a plurality or library of template nucleic acid molecules (e.g. genomic DNA fragments), using a single pair of universal forward and reverse primers, provided that each template molecule in the mixture is modified by the addition of the same universal primer-binding sequences.
Such “universal primer” approaches to solid-phase amplification are advantageous since they enable multiple template molecules of the same or different, known or unknown sequence to be amplified in a single amplification reaction on a solid support bearing “universal” forward and reverse primers.
The drawback of the standard “universal primer” approach is that hitherto it has been necessary to carry out several solution phase ligation reaction and purification steps on the targets in order to prepare the target-adaptor (or template) constructs suitable for amplification using the universal forward and reverse primers. Suitable template constructs, or libraries of template constructs, to be amplified with universal primers must be prepared by modifying the target polynucleotides that it is desired to amplify by addition of known adaptor sequences to the 5′ and 3′ ends of the target molecule(s). The target molecules themselves may be any polynucleotide molecules it is desired to amplify (e.g. random fragments of human genomic DNA). The adaptors are typically short oligonucleotides that may be synthesised by conventional means. The adaptors are usually attached to the 5′ and 3′ ends of target nucleic acid fragments by ligation in solution phase, prior to attachment of the template to a solid support, or by sub-cloning of the target into a vector at a cloning site that is flanked by suitable adaptor sequences.
All of the known template preparation methods require solution-phase reaction steps, after which the prepared template must be immobilised on a solid support such that solid-phase amplification can proceed.
The present inventors now describe a method of directly preparing template constructs for solid-phase amplification that reduces or removes the need for solution phase ligation reaction steps. The method involves the use of a ligation reaction to directly attach a first end of an unmodified target polynucleotide to the adaptor constructs immobilised on a solid support. The second end of the target polynucleotide can undergo a ligation reaction once immobilised, or can have been treated prior to the ligation of the first end. The method produces template construct(s) immobilised on a solid support that can subsequently be amplified by solid-phase amplification using multiple copies of a single primer-pair, or even a single primer. The method of the invention is applicable to whole-genome amplification as well as mono-template amplifications.